1. Field of the Invention
The invention pertains to methods and compositions for detecting anti-hepatitis E virus activity in a subject. The compositions include antigenic peptides of hepatitis E virus and mixtures of antigenic peptides of hepatitis E virus. The methods include serologic diagnosis of hepatitis E viral infection using the peptides and peptide mixtures of this invention.
2. Background Art
Hepatitis E virus (HEV) is a recently discovered agent of enterically transmitted non-A, non-B hepatitis (ET-NANB). The disease remains a serious problem in many developing countries. Unlike other agents of viral hepatitis, HEV infection is often associated with high mortality rates in infected pregnant women.
The first reported outbreak of ET-NANB hepatitis occurred in New Delhi, India in 1955. However, only after serologic tests for IgM anti-hepatitis A virus became available to exclude hepatitis A virus as the cause, was this very large outbreak recognized as ET-NANB hepatitis. Since that time epidemics of ET-NANB infection have been documented in many countries.
Until recently, the diagnosis of ET-NANB hepatitis outbreaks could only be based upon the absence of serologic markers of hepatitis A virus (HAV) and hepatitis B virus (HBV). Subsequently, specific tests for the detection of the ET-NANB hepatitis were based upon immune electron microscopy (IEM), in which a small volume of a stool suspension from acutely infected individuals is incubated with acute- or convalescent-phase sera and examined by electron microscopy (Bradley et al. PNAS USA 1987;84:6277-6281, 1987). IEM, thus identified 27-32 nm virus-like particles using acute and convalescent phase sera as the source of antibody. However, since most clinical specimens do not contain sufficient virus-like particles to visualize using IEM, this method is not useful for clinical or epidemiological analysis.
More recently, Reyes et al. (Science 247:1335-1339, 1990) successfully isolated and sequenced a partial cDNA clone from HEV. The HEV genome has subsequently been characterized as an RNA positive strand virus with an organization similar to Caliciviruses. Three open reading frames (ORF) have been identified (Tam et al. Virology, 185:120-131, 1991). Two type-common HEV epitopes were identified in proteins encoded by ORF2 and ORF3 (Reyes et al. Gastroenterologia Japonica 26 (suppl.3): 142-147, 1991b; Ichikawa et al. Immunol. 35:535-543, 1991). Both are localized at the C-terminus of their respective proteins. These epitopes were expressed as hybrid proteins with beta-galactosidase or glutathione-S-transferase and were recognized or an enzyme immunoassay?] by antibodies from acute- and convalescent-phase sera obtained from experimentally infected cynomologus macaques (Reyes et al., in "Viral hepatitis C,D,E", T. Shikata, R.H. Purcell, T. Uchida (Eds.) Elsevier Science Publishers, NY, pp.237-245, 1991a) or humans (Goldsmith et al., Lancet 339:328-331, 1992). These hybrid proteins have the disadvantage that the chimeric part of protein can negatively influence folding. Furthermore, individuals may have antibodies expressed to these sequences.
ORF2 has been suggested to be responsible for the expression of the HEV structural protein(s) (Tam et al., 1991). In addition, the recombinant polypeptide containing the C-terminal half of the protein has been shown to be an important diagnostic reagent for the detection of anti-HEV activity in patients infected with HEV.
Reyes et al. (l991a) demonstrated that a short fragment of the C-terminal region of the protein encoded by ORF3, obtained by expression of DNA derived from the HEV genome of the Burma strain did not react with sera from cynomologous macaques infected with the Mexico strain of HEV. Conversely, expressed recombinant protein derived from the Mexico strain did not react with sera from macaques infected with the Burma strain of HEV (Yarbough et al. J. Virol. 65:5790-5797, 1991). Sequence comparison of the two strains at the C-terminal region of ORF3 revealed a 78% homology (Yarbough et al., 1991). Furthermore, there appear to be type-common viral epitopes that are shared by divergent geographic isolates from Asia and North America (Yarbough et al. 1991; Goldsmith et al., 1992).
Thus, because of the lack of sensitivity and difficulty of performing the previously available tests, there exists a need for a rapid, simple and highly sensitive diagnostic test for HEV infection.
The present invention meets these needs by providing synthetic peptides and their use in a diagnostic test for the detection of antibodies to the hepatitis E virus. The present invention provides for the application of synthetic peptides in an immunodiagnostic assay for the detection of antibodies to HEV (anti-HEV).